The objectives of this proposal are to investigate microbial pathways of short-chain hydrocarbon metabolism and the properties of the enzymes, cofactors, and regulatory elements associated with these pathways. While hydrocarbon activation is interesting in its own right, perhaps even more interesting, and with broader implications for human health, are the nature and fate of highly toxic hydrocarbon oxidation products. For example, the ubiquitous oxidation of short-chain alkenes such as ethylene, propylene, styrene and isoprene (2-methybutadiene) results in the production of epoxides, which are highly electrophilic alkylating agents. The ubiquitous oxidation of short-chain saturated hydrocarbons involves either terminal or subterminal hydroxylation to produce primary or secondary alcohols, respectively. The further oxidation of these alcohols produces toxic aldehydes (from primary alcohols) or ketones (from secondary alcohols). Epoxides, aldehydes and ketones are also produced as intermediates or end-products of other metabolic pathways and by industrial processes. In spite of the toxicity of these compounds, a number of bacteria are able to grow using aliphatic epoxides, aldehydes and ketones as carbon and energy sources, either directly or indirectly (i.e. by forming them as intermediates of hydrocarbon catabolism). Work in our laboratory has revealed that novel transformations, cofactors and enzymes are involved in bacterial epoxide and ketone metabolism. Specifically, we discovered CO2-dependent pathways and enzymes for aliphatic epoxide and ketone metabolism, wherein epoxides and ketones are carboxylated to beta-keto acids for entry to central metabolism. In spite of the superficial similarities of these transformations, the mechanistic details of the carboxylation reactions are fundamentally different. We intend to further our work on the central roles of carboxylation and novel cofactors/enzymes in bacterial hydrocarbon/epoxide/ketone metabolism by pursuing the biochemical, structural, and genetic characterization of these processes.